Fanconi anemia (FA) is a major inherited bone marrow (BM) failure syndrome with extremely high risk of developing acute myeloid leukemia (AML). The only curable treatment for this devastating disease is stem cell and gene therapies through BM transplantation. We recently described cell-autonomous defects of FA hematopoietic stem cells (HSCs) in BM homing and engraftment, and showed that these impaired functions were associated with a decrease in the activity of the Rho GTPase Cdc42 known to be essential for cell polarity, adhesion and migration. These results provide the first evidence for a missing link between FA deficiency and inefficient HSC engraftment. More recently, our preliminary studies show that genetic deletion of Cdc42 in mice causes massive mobilization of HSCs from BM niche. Furthermore, we have developed CASIN, a Cdc42 activity-specific inhibitor, which can specifically, transiently, and reversibly down regulate Cdc42 and Cdc42-regulated signaling activities, allowing successful engraftment of CD34+ human blood progenitors as well as transplanted congenic murine HSCs without myeloablative or irradiation preconditioning. Using a FA knockout (Fanca) mouse model we have shown that preconditioning of Fanca-/- mice with CASIN allows significant engraftment of WT or FANCA gene-corrected Fanca-/- HSCs in the absence of myeloablative or irradiation conditioning. These preliminary data suggest that Cdc42 controls HSC residence in the BM niche through regulating cell adhesion. We hypothesize that cell-autonomous defect of FA HSC engraftment is a direct consequence of decreased Cdc42 activity, a property that could be utilized to vacant BM niche otherwise occupied by mutant HSC or leukemic stem cells (LSCs) and allow wild-type or gene-corrected HSC to engraft. The goals of the project are to study the mechanism of Cdc42 targeting in the context of HSC mobilization and BM niche engraftment, and ultimately to develop a novel preclinical regimen by targeting Cdc42 to improve stem cell and gene therapies for leukemia and BM failure diseases, clinical settings in which intensive preconditioning and scarce stem cell numbers critically limit success. The project presents the first mechanistic study aimed at targeting a critical HSC-niche interaction regulator in a significant health-care setting. The knowledge gained from the proposed study will not only improve mechanistic understanding of stem cell mobilization and engraftment in the context of stem cell transplantation, but also lead to a new avenue of research designed to target Cdc42 for developing innovative therapeutic regimens for stem cell and gene therapies in leukemia and BM failure diseases. PUBLIC HEALTH RELEVANCE: Bone marrow transplantation (BMT) is the only curable treatment for certain devastating blood diseases such as leukemia and bone marrow (BM) failure syndromes. However, three major hurdles that have been hampering scientific and clinical advance in the BMT field: 1) ineffective mobilization of patient stem cells;2) hypersensitivity of recipient patients to pre-conditioning regimens;and 3) inefficient delivery of genetically corrected patient stem cells to the BM niche. The goal of the project is to develop a novel preclinical regimen to improve BMT therapy for leukemia and BM failure diseases, clinical settings in which intensive preconditioning and scarce stem cell numbers critically limit success.